Human norovirus culture in B cells

MK Jones, KR Grau, V Costantini, AO Kolawole, Miranda de Graaf, P Freiden, CL Graves, Marion Koopmans, SM Wallet, SA Tibbetts, S Schultz-Cherry, CE Wobus, J Vinje, SM Karst

Research output: Contribution to journalArticleAcademicpeer-review

179 Citations (Scopus)

Abstract

Human noroviruses (HuNoVs) are a leading cause of foodborne disease and severe childhood diarrhea, and they cause a majority of the gastroenteritis outbreaks worldwide. However, the development of effective and long-lasting HuNoV vaccines and therapeutics has been greatly hindered by their uncultivability. We recently demonstrated that a HuNoV replicates in human B cells, and that commensal bacteria serve as a cofactor for this infection. In this protocol, we provide detailed methods for culturing the GII.4-Sydney HuNoV strain directly in human B cells, and in a coculture system in which the virus must cross a confluent epithelial barrier to access underlying B cells. We also describe methods for bacterial stimulation of HuNoV B cell infection and for measuring viral attachment to the surface of B cells. Finally, we highlight variables that contribute to the efficiency of viral replication in this system. Infection assays require 3 d and attachment assays require 3 h. Analysis of infection or attachment samples, including RNA extraction and RTRT-qPCR, requires similar to 6 h.
Original languageUndefined/Unknown
Pages (from-to)1939-1947
Number of pages9
JournalNature Protocols
Volume10
Issue number12
DOIs
Publication statusPublished - 2015

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